本文選題：中華絨螯蟹　切入點(diǎn)：糖原合酶激酶3　出處：《中國科學(xué)院大學(xué)(中國科學(xué)院海洋研究所)》2017年碩士論文

【摘要】：免疫系統(tǒng)持續(xù)地感知和應(yīng)答環(huán)境的威脅,需要消耗大量的能量,能量缺乏會(huì)導(dǎo)致免疫系統(tǒng)抑制和抗性降低,所以免疫反應(yīng)過程中能量的調(diào)節(jié)十分關(guān)鍵。PI3K/Akt信號(hào)通路過去被認(rèn)為是重要的代謝調(diào)節(jié)通路,在糖原代謝中發(fā)揮關(guān)鍵作用,近年來的研究發(fā)現(xiàn)PI3K/Akt參與了對(duì)于炎癥的調(diào)節(jié)。與PI3K/Akt關(guān)系緊密的RAS/ERK信號(hào)通路亦被證明對(duì)炎癥的發(fā)展起重要的調(diào)控作用。本研究以中華絨螯蟹(Eriocheir sinensis)為研究對(duì)象,利用分子生物學(xué)和生物信息學(xué)等手段,研究糖原合成酶激酶3以及其上游的兩種調(diào)控激酶蛋白激酶B(Akt)與細(xì)胞外信號(hào)調(diào)節(jié)激酶2(ERK2),探索PI3K與ERK信號(hào)通路在中華絨螯蟹固有免疫反應(yīng)中所發(fā)揮的作用。糖原合成酶激酶-3(GSK3)是催化絲氨酸/蘇氨酸的蛋白激酶,最早被鑒定為糖原合成的調(diào)控分子。本研究從中華絨螯蟹中克隆了GSK3同源基因(命名為EsGSK3)。其開放閱讀框長1824 bp,編碼一個(gè)具有607個(gè)氨基酸的蛋白質(zhì)。在EsGSK3中存在保守的絲氨酸/蘇氨酸激酶結(jié)構(gòu)域和DNA結(jié)合結(jié)構(gòu)域。EsGSK3首先與日本沼蝦(Macrobrachium nipponense)的GSK3-β聚類后納入無脊椎動(dòng)物分支,與來自脊椎動(dòng)物的GSK3s形成不同的分支。在中華絨螯蟹的肝胰腺、眼柄、肌肉、性腺、血細(xì)胞和造血組織中均可檢測到EsGSK3的mRNA表達(dá),其中在肝胰腺中的表達(dá)水平最高,且EsGSK3蛋白主要定位于血細(xì)胞的細(xì)胞質(zhì)中。EsGSK3 mRNA表達(dá)水平在嗜水氣單胞菌(Aeromonas hydrophila)刺激后6小時(shí)顯著升高(p0.05),在48小時(shí)后逐漸降至初始水平(p0.05)。脂多糖誘導(dǎo)的腫瘤壞死因子(TNF)-α因子(EsLITAF)的mRNA表達(dá)水平在嗜水氣單胞菌刺激后顯著升高。然而,利用特異性抑制劑鋰抑制EsGSK3的活性后,中華絨螯蟹肝胰腺中糖原水平顯著增加(p0.05),血淋巴上清中葡萄糖的含量顯著下調(diào),血淋巴細(xì)胞中的EsLITAF mRNA表達(dá)水平和血清中TNF-α含量均受到顯著抑制,而血淋巴細(xì)胞中核因子κB抑制劑(IκB)的mRNA表達(dá)水平顯著上升(p0.05),血細(xì)胞的吞噬率顯著升高,過氧化氫酶、堿性磷酸酶活性以及一氧化氮(NO)合成的增強(qiáng)。作為GSK3通路上游的激酶,細(xì)胞外信號(hào)調(diào)節(jié)激酶(ERK)被認(rèn)為是絲裂原活化蛋白激酶(MAPK)信號(hào)通路中一系列基因代謝的調(diào)節(jié)因子。從中華絨螯蟹cDNA文庫中鑒定出ERK的同源基因,命名為EsERK。其開放閱讀框全長為1746 bp,編碼581個(gè)氨基酸組成的多肽。分別分析了體內(nèi)EsERK的mRNA在受嗜水氣單胞菌刺激和dsRNA干擾后的表達(dá)變化。發(fā)現(xiàn)在嗜水氣單胞菌刺激6小時(shí)后,中華絨螯蟹血淋巴細(xì)胞中的EsERK mRNA表達(dá)水平顯著升高(p0.05),在48小時(shí)逐漸降低(p0.05)。對(duì)EsERK進(jìn)行RNA干擾24小時(shí)后,中華絨螯蟹肝胰腺中糖原水平顯著增加(p0.05),血淋巴上清中葡萄糖的含量顯著下調(diào),血淋巴細(xì)胞中核因子κB抑制劑(IκB)的mRNA表達(dá)水平顯著上升(p0.05),超氧化物歧化酶活性顯著升高(p0.05)。在抑制EsGSK3活性6小時(shí)后,中華絨螯蟹血淋巴細(xì)胞中EsIκB的mRNA表達(dá)水平顯著從中華絨螯蟹cDNA文庫中鑒定出蛋白激酶B(Akt)基因,并將其命名為EsAkt。其開放閱讀框全長為1395 bp,編碼一個(gè)具有464個(gè)氨基酸的蛋白質(zhì),其理論分子量為53.17 kDa,理論等電點(diǎn)為5.83�；赗T-PCR分析,在所有檢測的組織,包括血細(xì)胞,性腺,肝胰腺,鰓,肌肉和眼柄中均檢測到EsERK基因mRNA表達(dá),其中在肌肉的表達(dá)水平最高。在受到嗜水氣單胞菌(A.hydrophila)刺激12小時(shí)后,中華絨螯蟹血淋巴細(xì)胞中EsAkt的mRNA表達(dá)水平顯著升高(p0.05)。以上結(jié)果表明,EsGSK3可以通過誘導(dǎo)TNF-α產(chǎn)生,抑制血細(xì)胞吞噬作用,并調(diào)節(jié)免疫相關(guān)酶的活性及NO的合成調(diào)控中華絨螯蟹的固有免疫應(yīng)答反應(yīng)。EsERK可以通過調(diào)節(jié)IκB和TNF-α轉(zhuǎn)錄因子的表達(dá)參與免疫應(yīng)答,抑制它們的作用會(huì)對(duì)通過PI3K/Akt與Ras/ERK通路導(dǎo)致的炎癥反應(yīng)造成影響。此外EsAkt亦表現(xiàn)出對(duì)于菌刺激的響應(yīng)。本研究首次揭示了GSK3、ERK及Akt作為糖原代謝相關(guān)通路的關(guān)鍵調(diào)節(jié)分子,在調(diào)節(jié)中華絨螯蟹的固有免疫應(yīng)答中發(fā)揮作用,為深入研究PI3K與ERK信號(hào)通路在感染防治及炎癥控制中的作用提供了基礎(chǔ)。
[Abstract]:The immune system continues to perception and response to environmental threats, need to consume a large amount of energy, energy deficiency leads to a decrease in the suppression of the immune system and resistance, so the energy in the process of regulating the immune response is the key of.PI3K/Akt signaling pathway was regarded as an important metabolic pathway, plays a key role in glycogen metabolism, the study found that in recent years PI3K/Akt is involved in regulating inflammation. RAS/ERK signaling pathway has a close relationship with the PI3K/Akt proved to be an important regulatory role in the development of inflammation. In this study, the Chinese Mitten Crab (Eriocheir sinensis) as the research object, using molecular biology and bioinformatics methods, study of glycogen synthase kinase 3 and its upper reaches two regulation of protein kinase B kinase (Akt) and extracellular signal regulated kinase 2 (ERK2), to explore the PI3K and ERK signaling pathway in innate immunity against Chinese Mitten Crab We should take the role of glycogen synthase kinase. -3 (GSK3) is a serine / threonine protein kinase catalytic, was identified as the molecular regulation of glycogen synthesis. In this study, the homologous genes of GSK3 were cloned from Chinese Mitten Crab (named EsGSK3). The open reading frame of 1824 BP in length, encoding a a protein of 607 amino acids in EsGSK3. The existence of conserved serine / threonine kinase domain and DNA binding domain of.EsGSK3 first and m.nipponense (Macrobrachium nipponense) GSK3- beta after clustering into the invertebrate branch, formed different branches and GSK3s. In the spinal animal from Chinese mitten crab hepatopancreas, eyestalk and muscle, gonad, blood cells and hematopoietic tissue expression can be detected in EsGSK3 mRNA, the expression level in hepatopancreas is the highest, and the cytoplasmic EsGSK3 protein was mainly localized in blood cells in.EsGSK3 mRNA The expression level in Aeromonas hydrophila (Aeromonas hydrophila) 6 hours after stimulation was significantly increased (P0.05), in 48 hours after gradually reduced to the initial level (P0.05). Tumor necrosis factor induced by lipopolysaccharide (TNF) - alpha factor (EsLITAF) expression level of mRNA was significantly increased in Aeromonas hydrophila after stimulation. However, the inhibition of EsGSK3 by specific inhibitors of the activity of lithium, glycogen levels of Chinese mitten crab hepatopancreas significantly increased (P0.05), the content of glucose in haemolymph supernatant significantly reduced blood lymphocytes EsLITAF expression level of mRNA and serum TNF- content was obviously inhibited, while blood lymphocyte nuclear factor kappa the inhibitor of B (I K B) significantly increased the expression level of mRNA (P0.05), significantly increased the phagocytic rate of blood cells catalase, alkaline phosphatase activity and nitric oxide (NO) synthesis is enhanced. As the GSK3 upstream kinase, fine Extracellular signal regulated kinase (ERK) is considered to be the mitogen activated protein kinase (MAPK) signaling pathway in a series of regulatory factor gene metabolism. From Eriocheir sinensis cDNA library identified homologous gene ERK, named EsERK. and its ORF is 1746 BP, encoding a polypeptide of 581 amino acids. Analysis of EsERK in the expression of mRNA in affected by Aeromonas hydrophila stimulation and after dsRNA interference. Found that stimulating 6 hours in Aeromonas hydrophila, the expression level of Chinese mitten crab EsERK mRNA lymphocytes increased significantly (P0.05), decreased in 48 hours (P0.05) of EsERK. RNA interference after 24 hours, the glycogen level of Chinese mitten crab hepatopancreas significantly increased (P0.05), the content of glucose in haemolymph supernatant significantly reduced blood lymphocyte nuclear factor kappa B inhibitor (I K B) significantly increased the expression level of mRNA (P0.05), The activity of superoxide dismutase (P0.05) increased significantly. The inhibition of EsGSK3 activity after 6 hours, the expression level of Chinese mitten crab blood lymphocyte EsI kappa B mRNA significantly from Eriocheir sinensis cDNA library identified protein kinase B (Akt) gene, and named it the EsAkt. ORF 1395 BP, encoding a protein with 464 amino acids. The theoretical molecular weight of 53.17 kDa, the isoelectric point of 5.83. based on RT-PCR analysis, in all tissues examined, including blood cells, hepatopancreas, gill, gonad, muscle and eyestalk were detected EsERK gene expression of mRNA, one of the highest in the the expression level in muscle. By Aeromonas hydrophila (A.hydrophila) 12 hours after stimulation, the expression level of Eriocheir sinensis EsAkt lymphocytes in mRNA were significantly increased (P0.05). These results indicate that EsGSK3 can be induced by TNF- alpha, inhibiting blood cell endocytosis Apoptosis,.EsERK inherent immune response activity and NO synthesis regulation and regulate immune related enzymes of the Chinese mitten crab can participate in the immune response by regulating expression of I kappa B alpha and TNF- transcription factor, inhibiting their function will affect the inflammatory reaction caused by PI3K/Akt and Ras/ERK EsAkt also pathway. Show a response to bacteria stimulation. This is the first study to show the GSK3, ERK and Akt as a key regulator of glycogen metabolism pathways, plays a role in regulating innate immune response of Chinese mitten crab, provide the basis for further research on the role of PI3K and ERK signal pathway in the prevention and treatment of infection and inflammation control.

【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院海洋研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S945

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本文編號(hào)：1675093